PMCA screening of retropharyngeal lymph nodes in white-tailed deer and comparisons with ELISA and IHC

Chronic wasting disease (CWD) is a prion disease affecting cervids. CWD diagnosis is conducted through enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in retropharyngeal lymph nodes. Unfortunately, these techniques have limited sensitivity against the biomarker (CWD-prions). Two in vitro prion amplification techniques, real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA), have shown promise in detecting CWD-prions in tissues and bodily fluids. Recent studies have demonstrated that RT-QuIC yields similar results compared to ELISA and IHC. Here, we analyzed 1003 retropharyngeal lymph nodes (RPLNs) from Texas white-tailed deer. PMCA detected CWD at a higher rate compared to ELISA/IHC, identified different prion strains, and revealed the presence of CWD-prions in places with no previous history. These findings suggest that PMCA exhibits greater sensitivity than current standard techniques and could be valuable for rapid and strain-specific CWD detection.

One of the disadvantages of the PMCA technique includes the time-consuming visualization of products by western blot.To overcome this limitation, reduce the amount of sample needed for analysis, and increase throughput, we standardized dot blots for the analysis of the PMCA products.This is convenient considering the analysis of large animal cohorts as the one tested in this study (n = 1003).To achieve this, we checked dot blots for their specificity to discriminate PrP Sc -positive from PrP Sc -negative samples.In a first approach, we analyzed brains from healthy Tg1536 mice 71 (overexpressing deer prion protein) with and without PK treatment.As shown in Fig. 1A and B, PrP C was easily detected by this technique and signals completely disappeared after PK treatments.Then, we evaluated the discrimination of PMCA products derived from PrP Sc -seeded and unseeded reactions.Our results further confirmed the specificity of our dot blot system as all seeded PMCA reactions provided positive signals while unseeded samples were not visualized (Fig. 1C and D).
Regardless of the specificities observed in our dot blot system, we understand that the chances to obtain false positive results increase using this technique.In addition, dot blots do not allow the visualization of the classical electrophoretic mobility shift observed between PK -treated and -untreated samples that help to confirm the presence of PrP Sc .For that reason, all dot blot -positive PMCA products obtained from deer's RPLNs reactions were further confirmed for their PrP Sc content using western blots.As observed in Fig. 2, western blot revealed the presence of PrP Sc electrophoretic patterns in all PMCA positive samples, including positive controls and

Screening of a large cohort of RPLN specimens reveal the increased sensitivity of PMCA over ELISA and IHC
Considering the effectiveness of dot blots in assessing PrP Sc signals and the possibility to increase the throughput of PMCA screenings, we analyzed RPLNs from 1003 white-tailed deer collected in Texas.These samples were obtained from a depopulated captive facility (n = 229) and from free-ranging deer at different Texas counties (n = 774) (Fig. 3).All tested samples were previously evaluated by IHC and/or ELISA.Importantly, the CWDstatus of these samples were available to the researchers running the PMCA assay only after the screening was completed.The overall detection in captive and free-ranging samples by ELISA/IHC and PMCA is presented in Figs. 4 and 5.In the case of captive deer, we were able to detect the presence of CWD-prions in 12.6% of the samples (29/229) while ELISA/IHC provided positive results in 10.5% specimens (24/229) (Fig. 4).This show a superior, although modest, increase in detection for PMCA when compared with the currently approved diagnostic methods.
The increased CWD-prion detection potential of PMCA over ELISA/IHC was best appreciated in samples collected from free-ranging animals.There, PMCA identified 16 samples as CWD-positive, a higher number compared to the single positive specimen detected by the other diagnostic methods.It is important to note that most of the free-ranging positive samples were obtained from two counties with mandatory CWD testing requirements: Medina (n = 11) and Uvalde (n = 4) (Fig. 5).Both counties had CWD containment and surveillance zones established in response to detections of CWD in free-raging and captive animals.It is relevant to note that our PMCA screening identified a CWD-positive deer in Kinney county (Fig. 5) where no cases of CWD have been reported to date.However, due to the uneven number of samples tested in each county, the results of this screening cannot be used as indicators of CWD-positivity across counties.
It is important to mention that while screening the captive and free-ranging-derived samples, only one of the specimens (captive) providing positive results in ELISA/IHC was negative in PMCA.This is relevant, as these results support the specificity of our assay.The single sample providing positive ELISA/IHC results but negative outcomes by PMCA was tested three additional times after CWD-statuses were unveiled to the investigators.PMCA outcomes for this sample were maintained in all replicates.Additionally, and as mentioned previously, all PMCA samples that resulted in positive detection in dot blots were further confirmed for their PrP Sc content by western blots.In this study, all samples displaying positive signals in dot blots displayed the expected electrophoretic mobility shift after PK treatments (Fig. 2).Overall, these results demonstrate the increased CWD-prion detection potential of PMCA over IHC/ELISA, and likely RT-QuIC, in RPLN specimens of white-tailed deer.These results are summarized in Table 1.

Detection of different prion strains in the state of Texas
Prions can manifest in multiple strains, each one linked with specific molecular, pathological and infective properties 72,73 .Notably, multiple CWD strains have already been identified in naturally occurring cervid populations of North America and Europe [74][75][76][77] .The identification and study of CWD strains in nature is of capital importance to understand their effect within animal populations, and their interactions with the environment and other animal species.Differently from RT-QuIC, PMCA is able to faithfully propagate the molecular features of prion strains while maintaining their unique infectious properties 78,79 .In this study, we were able to 77.17% Free-ranging (n=774) 22.83% Captive (n=229) n=1,003    www.nature.com/scientificreports/discriminate two different electrophoretical mobilities in positive PMCA products (Fig. 6).Specifically, sample 68420 (blue arrow) from Medina county presented a lower electrophoretical mobility compared to other samples identified as CWD-positive in our screening (yellow and green arrows).This result was confirmed by running PMCA products after deglycosylation (facilitating the identification of changes in the single, unglycosylated PrP band).Importantly, the sample displaying this unique electrophoretic mobility (indicative of a different protein conformation, hence, a different prion strain) was obtained from a free-ranging animal.This suggests that different CWD prion strains are naturally present in wild Texas deer populations.

Discussion
Currently approved diagnostic methods for CWD include ELISA and IHC.These assays are usually applied to obex and lymph node tissues collected postmortem 36 .Multiple reports describe potential strategies to diagnose live animals using less invasive samples such as blood, urine, feces, saliva and skin 15,52,[80][81][82][83][84][85] .In all these studies, the diagnostic sensitivity is established by using the CWD-status information obtained postmortem through the above-mentioned approved methods.Considering the theoretical higher sensitivity of prion replication assays over ELISA and IHC, it is imperative to study the performance of these amplification techniques in previously tested lymph node tissues.At present, two studies comparing the efficacy of RT-QuIC to identify CWD-seeding activity in lymph node tissues from white-tailed deer have been communicated 57,58 .These studies reported similar results when using both set of techniques, preliminarily suggesting that RT-QuIC has no major advantages compared to ELISA/IHC for this specific sample type.These unexpected results could be caused by the composition of the biological matrix, as other biological samples have been shown to alter PMCA and RT-QuIC efficiencies 66,86 .Importantly, some reports suggest that PMCA and RT-QuIC have differential performances depending on the  type of sample being studied 66,82,83,86 .At present, the PMCA screening of white-tailed deer lymph node tissues collected from large cohorts has not been reported.To fill this gap in knowledge, we screened over 1000 RPLN specimens from free-ranging and captive white-tailed deer that were previously tested using ELISA/IHC.
Our study included 774 RPLN samples from free-ranging white-tailed deer, and 229 equivalent specimens from captive animals.In both cases, PMCA provided higher prion detection ratios compared with ELISA/IHC.Although the increase in detection was modest (20.83%) in captive animals, the identification of CWD-positive deer in free-ranging animals was considerably higher (approximately 16 folds).We believe that these differences are due to the uneven of CWD-prions for the animals in these two groups.Due to the animal density of deer in captive facilities, their exposure to the agent is expected to occur in both higher quantities and frequencies that will favor faster peripheral replication and tropism to lymphoid tissues.In natural environments, the exposure of CWD prions is expected to occur infrequently and in lower doses, increasing the times between prion exposure and clinical disease.The latter is expected to be linked with a slower progression in peripheral prion accumulation, a fact that will explain the lower diagnostic efficiency of ELISA and IHC.Although reasonable, these hypotheses will require further evaluation in different captive and free-ranging cohorts of animals, and using different prion detection methods.
Although our results suggest a superior performance of PMCA over RT-QuIC in RPLN screenings, several technical limitations still persisted.One of the main disadvantages of PMCA over RT-QuIC is the time that the former method takes to provide results 59 .One of the main sources for these extended processing times lies in the visualization of the PMCA products through western blotting 59 .To partially address this concern, this study included the standardization of dot blots for the visualization of PMCA products.One limitation of dot blots includes their potential to provide positive signals in samples that did not experienced a proper PK digestion.This could result in an increase of the rate of false positive results, compromising the overall detection system.Considering this, our dot blot system was controlled by multiple known positive and negative samples that supported our readings.To add rigor to our methodology, all PMCA products providing positive signals in dot blots were further evaluated by western blotting to visualize the specific electrophoretic mobility of PK digested PrP 79,87 .By using this methodology, no false positive signals were found for all CWD-positive PMCA products.This suggest that the use of dot blots may be useful to reduce processing times when handling a large number of samples.Nevertheless, the potential of false positive using PMCA and RT-QuIC is always a possibility.For that reason, experiments involving these assays need to be performed with caution by trained personnel and using appropriate positive and negative controls.We are confident that the positive results obtained in this screening are not due to contamination for several reasons.First, we used a large number of negative controls (unseeded PMCA reactions) across the experiment (n = 108, over 10% of the samples analyzed) and none of these controls provided positive signals.Second, our extensive experience using PMCA has shown that once a PMCA set is contaminated, a high number of positive reactions arise.This was not observed in this particular screening.Third, all the samples that were positive by IHC and/or ELISA, except for one, were also positive by PMCA (discussed below).
An additional positive outcome of this project includes the identification of different CWD-prion strains.Prions can manifest in different strains with the ability to differentially transmit within and across species 88,89 .For that reason, it is important to recognize and study potential strains operating in natural settings.Examples of the potential repercussions of prion strains lie in studies demonstrating that Norwegian deer and reindeer CWD prions are unable to infect humanized mice 90 .On the contrary, an in vitro study showed the potential of American deer prions to infect humanized mice after serial passaging, as well as other animal species 91 .One advantage of PMCA over RT-QuIC lies in the ability of the former to discriminate between prion strains 78,79 .This proved to be useful in our screening, as PMCA products with different electrophoretic mobilities after PK digestion were identified.Differences in electrophoretic mobility after PK digestion are considered as one of the gold-standard features to discriminate between prion strains.The properties of the different CWD prion strains identified in our screening are currently unknown, and whether they are equivalent to other previously identified CWD agents in white-tailed deer will be addressed in future studies.Current experiments in our laboratory are focused to characterize the identity of this particular CWD prion variants in terms of their biochemical, pathological and infectious properties.Future PMCA screenings in larger animal cohorts collected from geographically diverse sites may unveil the presence of additional, naturally occurring strains operating in captive facilities and in the landscape.
As mentioned above, PMCA was able to identify all but one of the CWD-positive RPLNs specimens detected by IHC.This outcome could be due to multiple reasons, including the following: (i) PMCA is unable to detect specific naturally-occurring prion strains, (ii) the uneven distribution of PrP Sc deposits in this tissue, (iii) the sample provided a false positive signal in IHC/ELISA, or (iv) a sampling error.In order to address the former, this sample is currently being investigated by other prion detection methods, including RT-QuIC and bioassays.Future studies will provide clarity on whether PMCA is able to detect only a limited array of CWD prion isolates.
Although this study provides relevant evidence useful for CWD prion detection, some limitations are also identified.One of them involves the regional limitation of our sample.The incidence of CWD in Texas is low compared with other states 32,92 .Considering this, the samples tested in this study may not be representative of the whole CWD spectrum, and future studies analyzing larger and diverse cohorts may be needed to confirm our results.In addition, this experiment is restricted to white-tailed deer, one of the many animal species affected by CWD 32,[92][93][94][95][96] .To address this problem, future studies considering mule deer, elk, and other species should be performed.Finally, other sample types should also be considered in future studies to identify potentially relevant specimens that could be used for diagnostic purposes.
Overall, our study provides relevant information for the detection of CWD prions by comparing established techniques with a prion replication method.As expected, our results demonstrate the higher diagnostic potential of PMCA over the currently approved diagnostic techniques when using the same sample type.Moreover, we

Sample collection and preparation
Retropharyngeal lymph nodes (RPLN) from free-ranging and captive white-tailed deer were donated by hunters or collected by Texas Parks and Wildlife Department (TPWD) personnel from breeding premises.These samples were stored at − 20 °C until shipped to UTHealth facilities where they were also stored at − 20 °C.Each sample was stored in an individual bag for free range samples and in double bags for captive samples.Specimens were partially thawed in a disposable container and a piece of tissue (0.2 g) was collected using disposable forceps and blades.The disposable container, blades and tweezers were replaced after use (in contact with a single sample).In addition, manipulator's gloves were replaced at least once when handling a single sample.The use of disposable equipment and personal protective equipment (PPE) was critical considering the ultrasensitive nature of PMCA that facilitates cross-contamination.Tissues were homogenized at 20% w/v in PBS containing a cocktail of protease inhibitors (Roche, Basel, Switzerland) using a Precellys 24 dual homogenizer (Bertin instruments, MP biomedicals, Irvine, CA, USA).

Protein misfolding cyclic amplification (PMCA)
The PMCA process was carried out as reported in detail in our prior publications 59 and optimized for CWD 67 .In short, PMCA substrate was prepared from Tg1536 mice's brains homogenized at 10% w/v in PMCA conversion buffer (150 mM NaCl, 1% Triton X-100, and a protease inhibitors cocktail (Roche, Basel, Switzerland) in PBS).Ninety µL aliquots of substrate were combined with 10 µL of RPLN homogenates (equivalent to 2 µg of wet tissue) or positive control brain extracts.The first PMCA round consisted of 144 incubation/sonication cycles, while the second and third rounds included 96 cycles each.Serial PMCA rounds were performed by mixing 10 µL of PMCA products with 90 µL of fresh PMCA substrate.Each PMCA cycle consisted of 29 min and 40 s of incubation, and 20 s of sonication.Each PMCA set included reactions supplemented with serial dilutions of a CWD brain of known seeding activity, as described 65 .Negative controls included four unseeded reactions per PMCA set.Results were assessed at the third PMCA round.It is important to consider that all unseeded PMCA reactions included in our assay (n = 108) resulted in negative PrP Sc signals.The positive control PMCA reactions (as shown in Fig. 1) were prepared by mixing PMCA substrates with brain extracts from a terminally ill Tg1536 mouse infected with CWD (10 -5 dilution).

Proteinase K (PK) treatment
To visualize the content of the protease-resistant prion protein present in the PMCA products, samples were treated with PK (100 µg/mL final concentration, Sigma-Aldrich, Saint Louis, MO, USA).The conditions used for PK digestion included 37 °C with shaking at 450 rpm (in an Eppendorf thermomixer) for 80 min.PK reactions were stopped by the addition of either phenylmethylsulphonyl fluoride (Sigma-Aldrich, Saint Louis, MO, USA) at a final concentration of 5 mM and heating (70 °C for 10 min) for dot blot analyses, or LDS sample buffer (Fisher Scientific, Walthmam, MA, USA) and heating (95 °C for 10 min) for western blotting.

Dot blotting
To visualize PK-resistant PrP, 5 µL of PK-digested PMCA products were placed in a nitrocellulose membrane (GE Healthcare Amersham, Chicago, IL, USA) using a Bio-dot apparatus (Bio-Rad Laboratories, Hercules, CA, USA), following manufacturer's recommendations.The membranes were dried using a hairdryer and placed into an incubation chamber containing 3 M guanidinium hydrochloride for 10 min.The membrane was rinsed using washing buffer (1X PBS and 0.05% Tween20 (Sigma Aldrich, Saint Louis, MO, USA)), then incubated with 5% w/v non-fat milk solution and probed with monoclonal purified 6D11 antibody (Biolegend, San Diego, CA, USA) at 1:10,000 dilution.Membranes were washed using washing buffer (0.05% v/v of Tween20 (Sigma Aldrich, Saint Louis, MO, USA) in PBS) and then incubated with a polyclonal anti-mouse IgG (whole molecule)-peroxidase antibody produced in sheep (Sigma-Aldrich, Saint Louis, MO, USA) at a 1:3000 dilution.Membranes were washed and developed using ECL (GE Healthcare Amersham, Chicago, IL, USA) following manufacturer's recommendations.Samples providing positive PK-resistant PrP signals were subsequently run by western blot to rule out the presence of false positives.

Western blotting
For western blots, a similar procedure as dot blot was performed with the difference that PK-digested samples were fractioned in NuPAGE 12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA) and then transferred to nitrocellulose membranes (GE Healthcare Amersham, Chicago, IL, USA), as previously reported 67,69 .Membranes were blocked using 5% w/v non-fat milk solution and probed with monoclonal purified 6D11 antibody (Biolegend, San Diego, CA, USA) at a 1:10,000 dilution.Besides of the step involving incubation with the guanidinium hydrochloride solution, the rest of the process was the same as described for dot blotting.

Protein deglycosilation assay
To study the presence of different electrophoretic mobilities in PK-digested PrP Sc , positive PMCA products were subjected to deglycosylation with glycerol-free N-glycosidase (New England BioLabs, Ipswich, MA, USA).Briefly, 50 µL of sample were digested with 50 µg/mL of PK (final concentration) for 1 h at 37 °C and then centrifuged at 100,000×g for 1 h at 4 °C.The resulting pellets were resuspended in 50 µL of denaturant buffer (New England BioLabs, Ipswich, MA, USA) and heated for 10 min at 95 °C.Then, 6.5 µL of NP40 buffer, 6.5 µL of 10X deglycosilation buffer and 1 µL of Peptide-N-Glycosidase F (PNGase F) solution were added to the sample followed by overnight incubation at 37 °C with shaking at 450 rpm in an Eppendorf thermomixer.Finally, 25 µL of 4X LDS sample buffer were added to the mixture and western blot analysis was performed as described above.The membranes were probed with monoclonal purified 6D11 antibody (Biolegend, San Diego, CA, USA) at a 1:10,000 dilution.

Figure 1 .Figure 2 .
Figure 1.Representative images depicting the visualization of PMCA products by dot blots.(A) Scheme depicting the position of PK-digested (white dots) and untreated (black dots) brain extracts from an uninfected (PrP Sc -free) Tg1536 mouse.(B) Dot blot depicting PK-digested and untreated brain extracts from an uninfected (PrP Sc -free) Tg1536 mouse placed in a membrane following the scheme shown in (A).(C) Scheme depicting the position of CWD-seeded (red dots) and unseeded (green dots) PMCA products.(D) Dot blot results from CWD-seeded and unseeded PMCA products following the plan shown in (C).All samples in C were PK treated as explained in Materials and Methods.The CWD prion "seed" used in this experiment correspond to the brain extract of a terminally ill tg1536 mouse (expressing the deer prion protein).This mouse was intracerebrally inoculated with the brain extract of a terminally ill, experimentally infected white-tailed deer (PrP 96GG, kindly donated by Dr. Edward Hoover).

Figure 3 .
Figure 3. Distribution and origin of the white-tailed deer RPLN samples analyzed.(A) Distribution of samples considering their habitats (captive or free-ranging).(B) Distribution of free-ranging samples by Texas county.

Figure 4 .Figure 5 .
Figure 4. Summary of the PMCA-screening in 229 RPLN specimens collected from captive and free-ranging Texas white-tailed deer.Comparison of CWD status for RPLN specimens in captive and free-ranging deer by IHC/ELISA (red) and PMCA (blue).The ratios depicted at the top of each column represent the number of animals identified as CWD-prions carriers by each technique versus the total number of animals analyzed.Percentual values of detection by each technique are also provided.

Figure 6 .
Figure 6.Electrophoretic mobility differences of selected PMCA products derived from RPLNs of free-ranging Texas white-tailed deer.(A) Electrophoretic mobilities of PMCA products from four positive and one negative PMCA products.(B) Electrophoretic mobilities of PMCA products from four selected positive PMCA products after PNGase treatment.Membranes were probed with the 6D11 antibody.All samples shown in this figure have been PK-treated.Numbers at the top of each panel are arbitrary and depict identification codes.Colored arrows indicate the same samples in both panels.Numbers at the left of blots depict molecular weight markers.Raw data is provided as Supplementary Information.

Table 1 .
Comparison of IHC/ELISA and PMCA CWD-prion detection considering the habitat of white-tailed deer included in this study.Fold-change increases in PMCA detection over IHC/ELISA is depicted in the right-end column.